There is a considerable amount of prior art regarding the immobilization of E. coli or other microbial cells for use in the preparation of L-aspartic acid. For example, U.S. Pat. No. 3,791,926 (Chibata et al) describes a process for the production of L-aspartic acid which involves polymerizing a monomer selected from acrylamide, N,N'-lower alkylene-bis(acrylamide) and bis(acrylamidomethyl) ether in an aqueous suspension containing an aspartase-producing microorganism such as E. coli ATCC No. 11303. The resultant immobilized aspartase-producing microorganism is treated with ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt which by enzymatic reaction gives L-aspartic acid.
The immobilization of E. coli cells containing aspartase activity and use of the resulting immobilized cells for the production of L-aspartic acid are also described by Fusee et al, Applied and Environmental Microbiology, Vol. 42, No. 4, October 1981, pages 672-676. According to Fusee et al, the cells are immobilized by mixing a suspension of the cells with a liquid isocyanate-capped polyurethane prepolymer (Hypol.RTM.) so as to form a "foam" containing the immobilized cells.
Sato et al (Biochimica et Biophysica Acta, 570(1979) pages 179-186) have disclosed the immobilization of E. coli cells containing aspartase activity with .kappa.-carrageenan, and use of the immobilized preparation for the production of L-aspartic acid.
Additional literature disclosures describing the immobilization of microbial cells in urethane prepolymers or polyurethanes or the like include the following:
(a) Immobilization of Microbial Cells in Polyurethane Matrices by Klein et al, Biotechnology Letters, Vol. 3, No. 2, 65-70 (1981); PA0 (b) Hydrophilic Urethane Prepolymers: Convenient Materials for Enzyme Entrapment, Biotechnology & Bioengineering, Vol. XX, pages 1465-1469 (1978); PA0 (c) Transformation of Steroids by Gel-Entrapped Cells in Organic Solvent by Omata et al, European J. Applied Microbiology and Biotechnology 8, 143-155 (1979); and PA0 (d) Entrapment of Microbial Cells and Organelles With Hydrophilic Urethane Prepolymers, by Tanaka et al, European J. Applied Microbiology and Biotechnology, 7, 351-354 (1979). PA0 (a) In the case of the preferred polyazetidine prepolymers, E. coli ATCC 11303 cells are advantageously mixed with an aqueous solution of the prepolymer so as to obtain a homogeneous mixture after which the polyazetidine may be cured or crosslinked to give an insoluble L-aspartase active composition by any of the following means: PA0 (b) For carboxymethyl cellulose (CMC) gums, homogeneous mixtures with E. coli ATCC 11303 may be crosslinked (cured) to insoluble, L-aspartase active coatings, membranes, fibers, beads, etc., by contacting an aqueous mixture of, for example, 1.0 part/wt. of cell paste/CMC mixture with an aqueous solution of 0.001 to 1.0 parts/wt. of polyvalent cation salt or, ideally 0.01 to 0.4 parts/wt. of polyvalent cation salt per 1.0 part/wt. of the cell paste/CMC mixture. PA0 (c) When polyurethane hydrogels are used, homogeneous aqueous dispersions of E. coli ATCC 11303 (typically 1.0 g of cells per 1.0 to 1000 ml H.sub.2 O broadly and 10 to 100 ml H.sub.2 O preferably) are mixed with the hydrogel prepolymer and the prepolymer is cured into a water-insoluble L-aspartase active composition by allowing the polyisocyanate to react with water or in a more rapid manner by either removing 50 to 100% of the available water at 1000 to 0.1 Torr and below 50.degree. C. (preferably 760 to 5.0 Torr and 0.degree. to 30.degree. C.) or by exposing the undried compositions to aqueous solutions of molecules having more than two primary or secondary amine groups. Typically these polyamine molecules can range from hydrazine or ethylene diamine to polyethylene imine. Generally for each 1.0 g of hydrogel prepolymer used, from 0.01 to 10.0 g of the polyamine in from 1.0 to 500 ml of water could be used. Ideally 0.1 to 1.0 g of the polyamine and 10 to 100 ml of water are employed per gram of hydrogel prepolymer used. PA0 (i) A batch type process wherein the catalyst compositions are stirred in from 0.1 to 5.0 molar (preferably 0.5 to 2.0 molar) solutions of ammonium fumarate in water at 5.0 to 10.0 pH (preferably 7.5 to 9.5 pH) for periods of 1.0 to 100 hours (preferably 8 to 48 hours) at temperatures below 50.degree. C. (preferably 20.degree. to 40.degree. C.). Broadly from 0.05 to 50 g of immobilized cells, preferably from 1.0 to 15 g, are used per 1.0 mole of starting ammonium fumarate. After the conversion, the catalyst compositions may be removed by filtration or the equivalent for reuse in converting fresh batches of fumarate solutions. The product solutions are obtained in a form suitable for conventional processing to isolate the L-aspartic acid (acidification, precipitation, filtration, washing, recrystallization, drying). PA0 (ii) A continuous process wherein the catalyst compositions, e.g. coated beads, are placed in columns and the solutions of ammonium fumarate (concentrations, pH's, and temperatures are the same as described above for the batch processes) are passed through the catalyst beds either from above or from below (fluidized bed mode). The rates of passage of these fumarate solutions may range from 0.1 to 1000 space velocities/hour. For example, 5.0 liters of solution per hour may be passed through 1.0 liter of catalyst bed representing 5.0 space velocities (S.V.) per hour. Preferably the fumarate solution flow rates which yield essentially 100% conversion of the fumarate to the L-asparate fall in the range of 0.5 to 20.0 S.V./hour. The effluent from these columns of catalyst beds is suitable for conventional processing to isolate L-aspartic acid (as outlined above in the batch processes).
The above-noted processes for preparing L-aspartic acid using immobilized microbial cells suffer from various disadvantages. For example, .kappa.-carrageenan gum and polyurethane "foam" as disclosed by Fusee et al and Sato et al are relatively soft and compressible. Hence when these immobilized cell compositions are used, in a column through which ammonium fumarate is passed for conversion to ammonium aspartase, they tend to be compressed and plug up, particularly where high flow rates and/or relatively tall column heights are involved.